The major objectives of this study is to elucidate the lineage relationships between different cell types and different regionalized populations of cells in the developing limb, to establish patterns of cell movement and migration within the limb primordium, and to determine how lineage and migration are integrated with patterning signals to set the stage for limb morphogenesis. In spite of two decades of intense study on a molecular level, many critical aspects of limb patterning are still not understood. For example, it is not known why the limb bud forms in the place and time that it does. This process involves specific localized epithelial- mesenchymal transition (EMT), but how this is controlled to initiate limb bud formation has not been described. There also have been no studies clarifying why the limb bud grows out as a paddle, flattened along its dorsal- ventral axis, rather than as a hemispherical mound. Moreover, although the normal lineage relationships between different cell types within the limb bud are largely understood, the degree of plasticity in cell differentiation during, for instance, regenerative growth remains controversial. To address these issues we will utilize a range of state-of-the-art approaches including live imaging of cell movements by two-photon microscopy and lineage analysis using tagged libraries of retroviral vectors. Clues to cell behavior identified in these studies will be used to design functional studies of candidate genes that we hypothesize may be involved in regulating cell movements in the limb bud. For example, preliminary data shows that the dynamic expression of different Hox genes correlate with the timing of cells migrating into the limb from the lateral plate. Similarly, there is suggestive evidence that the activity of the secreted ligand Wnt5a plays a role in directing the cell movements leading to flattening of the limb bud into a paddle. Misexpression of wild type, activated, and dominant-negative forms of these genes in conjunction with imaging and analysis of cell migration and of lineage changes, will allow these hypothesis to be directly assessed.